Nucleic acids have been found to be useful analytes for the determination of the presence or absence of genes or microorganisms in human body fluids, food or environment in the field of health care. Nucleic acid analysis has found widespread use after the introduction of nucleic acid amplification, like the Polymerase Chain Reaction (PCR, see U.S. Pat. No. 4,683,202). Thus, sufficient amounts of nucleic acids are available from each sample. The nucleic acids can be determined from this pretreated sample using a variety of different techniques, dependent on the particular purpose. Most assays require the use of a probe which is either immobilized or immobilizable or is labelled by attachment of one or more reporter groups.
A reporter group has the characteristics to be itself capable to be determined or it can be reacted with reagents that make the probe determinable via said reporter group. Thus, for example, probes that are labelled by reporter groups can be determined, as can be hybrids that contain the probe and a nucleic acid to be determined. In case of immobilized probes, the hybrid between the probe and the nucleic acid to be determined is determined at the solid phase to which the probe is bound. In a particular form of assays, not only one nucleic acid having a specific sequence, but a large number of nucleic acids of different sequence is determined. For this purpose, the probes are immobilized in tiny spots in an array on a flat surface such as a glass chip (EP-A-0 476 014 and TIBTECH (1997), Vol. 15, 465-469, WO89/10977, WO89/11548, U.S. Pat. No. 5,202,231, U.S. Pat. No. 5,002,867, WO 93/17126). Further development has provided methods for making very large arrays of oligonucleotide probes in very small areas. (U.S. Pat. No. 5,143,854, WO 90/15070, WO 92/10092). Microfabricated arrays of large numbers of oligonucleotide probes, called “DNA chips” offer great promise for a wide variety of applications (see e.g. U.S. Pat. No. 6,156,501 and U.S. Pat. No. 6,022,963).
However, nucleic acid determinations often suffer from the problem that the base pairing possibilities between the natural bases A and T and C and G have different stability. This can be attributed to the different capability of these bases to form hydrogen bonding. Thus, the dA-dT-base pair has two hydrogen bridges, while the dG-dC-base pair has three hydrogen bridges. This results in different melting temperatures (Tm) of hybrids, depending on the GC content [1-3]. The higher the GC content, the higher the Tm. The hybridisation strength or the degree of hybridization may be investigated by the measurement of the Tm of the resulting duplex. This can be done by exposing a duplex in solution to gradually increasing temperature and monitoring the denaturation of the duplex, for example, by absorbance of ultraviolet light, which increases with the unstacking of base pairs that accompanies denaturation. The Tm is generally defined as the temperature midpoint of the transition from a fully duplex structure to complete denaturation, i.e. the formation of two isolated single strands.
Therefore in routine nucleic acid analysis, there is often the wish to change the Tm of a nucleic acid molecule. For example, for certain purposes it may be advantageous to equalize or harmonize the Tm of nucleic acids of the same length or to make it even independent from the length of the nucleic acid or the binding region in order to be in the position to apply similar hybridization conditions for all assays. This is particularly necessary for assays using arrays, as on such arrays the hybridizing conditions for each probe must be identical. One solution was the use of low hybridization temperatures. Under such conditions, many nucleic acids having a low degree of base sequence complementarity will bind to the probe. This is called unspecific binding which does not allow discrimination between similar sequences. Another proposal was directed to the use of chemical reagents in the hybridization mixture, for example the addition of tetramethylammonium chloride (TMAC). This reagent reduces the difference between the stability of dG-dC and dA-dT base pairs but the effect is insufficient for short oligonucleotides. Further the addition of salts such as TMAC may not be appreciated as it complicates the optimization of the assay. Another proposal was directed to the use of different concentrations of each different (immobilized) probe in one assay. This was found to be technically complex if not impossible on a chip surface. As a further option the substitution of ribonucleotides in an oligonucleotide composed of deoxyribonucleotides, and vice versa was applied for the adaptation of DNA stability, Hoheisel (1996), Nucleic Acids Res. 24, 430-432.
However, it may be also advantageous to increase the Tm of a given nucleic acid. This is interesting in the field of nucleic acids used for antisense therapy, mismatch discrimination and for nucleic acids used in diagnostics. The nucleic acids may be used as primers or probes. The aim is to allow a more simple design of primers and probes used in multiplex reactions and to synthesize shorter capture probes used on chips, as the chemical synthesis of oligonucleotides on a chip surface used for arrays is not as effective as in routine oligonucleotide synthesis. The relative contribution of each base pair to the melting temperature of a hybrid is the higher the shorter an oligonucleotide is. In consequence, the difference in stability between a mismatch and a perfect match is higher for shorter oligonucleotides. However, short oligonucleotides hybridize weakly and, therefore, the hybridization reaction has to be performed at low stringency. In consequence, the potential higher ability of discrimination between different sequences by shorter oligonucleotides can only be used under conditions of low stringency. It would be of considerable advantage to provide bases which allow to achieve a high level of mismatch discrimination under stringent conditions, in particular for short oligonucleotides at temperatures used e.g. in amplification reactions. Further, there is the desire in the state of the art to use short oligonucleotides with high discriminatory power in arrays as the chemical synthesis of oligonucleotides on solid supports used for arrays is not as effective as in routine synthesis. Therefore, the ability to use shorter oligonucleotides under stringent conditions would be of considerable advantage. If bases are found that lead to an increase of the Tm of an oligonucleotide hybridized to its complementary strand, other bases may then be used in the same oligonucleotide to further adjust the Tm according to the preferences of the test system to be used.
Theoretically, oligonucleotide duplexes forming other tridentate base pairs should exhibit a similar or higher stability, e.g. those with 2-aminoadenine opposite to thymine. Nevertheless, it has been shown that 2-aminoadenine-thymine/uracil base pairs exhibit only a low thermal stability [4-10]. From the data published so far one can conclude that the additional NH2-group of 2′-deoxyadenosin-2-amine (molecule 1 (see below); n2Ad) contributes very little to the base pair stability of a DNA duplex. The Tm-increase is in the range of only 1-2° C. Furthermore, this stabilization does not correspond to the total number of n2Ad-residues incorporated in the duplex instead of dA [11]. A stronger stabilization as reported for duplex DNA is found for duplex RNA or for DNA-RNA hybrids [9] [10] [12]. A rather high base pair stability is observed when 2-aminoadenine is introduced into PNA [13] or hexitol nucleic acids [14]. Modified backbones other than of DNA or of RNA appear to enhance the stability of the 2-aminoadenine-thymine/uracil pair.
The unusual behavior of oligonucleotide duplexes containing n2Ad-dT residues is interesting for the development of an adenine-thymine recognition motif showing the same or even a higher stability than a guanine-cytosine base pair. In the following compounds the purine moiety of compound 1 is replaced by an 8-aza-7-deazapurine (pyrazolo[3,4-d]pyrimidine) or a 7-deazapurine (pyrrolo[2,3-d]pyrimidine) leading to nucleosides (2a [15], 2b, 2c or 3 [16], [17] see below).

Compounds of similar chemical architecture were investigated in the prior art. The synthesis of 7-substituted-7-deaza and 8-aza-7-deazapurine 2′-deoxyribonucleotides, their incorporation into oligonucleotides, and the stability of the corresponding duplexes has been investigated (Seela et al. (1997) Nucleosides & Nucleotides 16, 963-966). This document does not contain a disclosure of 7-substituted 7-deaza-8-aza-diamino-purines. Stabilization of duplexes by pyrazolopyrimidine base analogues have been reported (Seela et al. (1988) Helv. Chim. Acta 71, 1191-1198; Seela et al. (1988) Helv. Chim Acta 71, 1813-1823; and Seela et al. (1989) Nucleic Acids Res. 17, 901-910)
Pyrazolo[3,4-d]pyrimidine residues in oligonucleotides are also useful as sites of attachment of various groups (WO90/14353). Oligonucleotides having incorporated one or more pyrazolo[3,4-d]pyrimidine have an enhanced tendency to form triplexes (Belousov et al. (1998). Nucleic Acids Res. 26, 1324-1328).
The compounds 7-iodo, 7-cyano and 7-propynyl-7-deaza-2-amino-2′-deoxyadenosine were synthesized by Balow et al. (1997, Nucleosides & Nucleotides 16, 941-944) and incorporated into oligonucleotide sequences. These oligonucleotides exhibit enhanced binding affinities to RNA complements relative to unmodified sequences. However, no corresponding 8-aza-compounds were made and investigated. Seela et al. (1999, Nucleosides & Nucleotides 18, 1399-1400) disclose 7-substituted 8-aza-7-deazapurine DNA, its synthesis and duplex stability. The authors do not address possible uses of the disclosed compounds.
WO 90/03370 discloses 3,4-disubstituted and 3,4,6-trisubstituted pyrazolo-[3,4-d]-pyrimidines, more particularly 4,6-diamino-pyrazolo-[3,4-d]-pyrimidines with a linker at the C3-position to which an intercalator, an electrophilic cross linker or a reporter group is attached. These compounds may be attached to sugars or incorporated into oligonucleotides and thereby used for the identification, isolation, localization and/or detection of complementary nucleic acid sequences of interest. U.S. Pat. No. 5,594,121 discloses novel oligomers with enhanced abilities to form duplexes or triplexes. The oligomers may contain 7-substituted 8-aza-7-deaza-diamino-purines with propinyl and aryls as substituents at the 7-position. Compositions containing these oligomers may used for diagnostic purposes.
There is still a need to provide probes with a high discriminatory power and with a short length, the Tm of which is high under stringent conditions and which can be used in various methods useful in the field of diagnostics as e.g. in the Lightcycler® system (Roche, Mannheim, Germany), TaqMan® (WO92/02638 and corresponding U.S. Pat. Nos. 5,210,015, 5,804,375, 5,487,972) or other applications involving fluorescence energy transfer.